There has been a growing level of activity in the development of therapeutic technology in recent years, and attempts at the clinical application thereof have been made at numerous institutions both in Japan and overseas. Various methods have been developed for this purpose, including methods consisting of the in vitro culturing of cells harvested from the body of a patient followed by re-transplanting the cells back into the patient, methods consisting of introducing a certain specific gene into harvested cells followed by returning the cells to the body, and transplantation methods consisting of seeding cells on a cell scaffold to construct three-dimensional structures. These technologies are used as medical technologies in the field of regenerative medicine, and have the potential to be able to treat diseases that were difficult to cure completely with conventional treatment. Consequently, the earliest possible practical application of effective cell-based therapeutic technologies is earnestly awaited so as to allow these technologies to be applied to numerous diseases.
Cells isolated from tissue or blood harvested from the body of a patient per se (autologous cells), cells isolated from tissue or blood harvested from the body of another person (heterologous cells), or established cells and the like are used in the field of regenerative medicine. The type of cell is suitably selected according to the disease site and pathology. Rejection reactions attributable to immunity present problems when transplanting cells. In the case cells different from one's own cells (heterologous cells) are transplanted, normally the type of major histocompatibility complex (MHC) does not match, resulting in the onset of a rejection reaction that prevents the cells from grafting.
On the other hand, in the case of using autologous cells, there is no occurrence of rejection reactions since the MHC types match by virtue of using the patient's own cells. Consequently, although regenerative medicine using autologous cells is preferable from the viewpoint of immunorejection, in the case of using autologous cells, since cells or tissue are harvested for each patient, the cells require treatment such as so-called “order-made” culturing resulting in exorbitant costs. On the other hand, treatment using heterologous cells would make it possible to ensure the required cells or tissues in advance and heterologous cells retained in storage could be used when a transplant was required, thereby making the use of heterologous cells preferable from the viewpoint of reducing costs. In this case, immunosuppressants can be used to avoid immunorejection in order to prevent the transplanted heterologous cells from being subjected to immunorejection within the body of the recipient.
Some of the cell types used in regenerative medicine either do not divide or grow or hardly grow at all in in vitro culture environments. Cardiomyocytes and nerve cells fall into this category, and it has been difficult to use such cells in regenerative medicine. However, with the discovery of pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS), even in the case of cells that do not divide or grow in the manner of cardiomyocytes and nerve cells, these cells have been able to be produced by inducing to differentiate from pluripotent stem cells, enabling these cells to be provided for use in regenerative medicine.
Pluripotent stem cells such as ES cells or iPS cells have the property of being able to differentiate into nearly all cells that compose the body. In addition, since ES cells or iPS cells, which retain the ability to remain undifferentiated prior to differentiation, have a self-replicating ability, they can basically be grown infinitely. Consequently, even in cases requiring large numbers of cells, pluripotent stem cells can theoretically be grown to the required number of cells. It has thus become possible to obtain the required number of cells by inducing pluripotent stem cells obtained in this manner to differentiate into any somatic cell.
Although ES cells and iPS cells can be grown in infinite numbers and can be induced to differentiate into any somatic cell, when cells still retaining the ability to remain undifferentiated are transplanted into the body, there is the risk of these cells forming a teratoma, which is a type of tumor, due to the pluripotency thereof. Although such problems do not occur if undifferentiated ES cells or iPS cells do not remain in populations of somatic cells that have been induced to differentiate from ES cells or iPS cells, if these ES cells or iPS cells that are still undifferentiated end up being transplanted into the body while remaining within a somatic cell population, there is the risk of the formation of a teratoma, thereby resulting concerns over safety (Non-Patent Document 1). Consequently, there is a need for a technology that reduces the number of undifferentiated pluripotent stem cells present in cell populations that have been induced to differentiate from pluripotent stem cells.
In order to overcome these problems, methods using a cell sorter (Non-Patent Document 2), methods incorporating suicide genes (Non-Patent Document 3) and methods using chemical inhibitors (Non-Patent Document 4, Non-Patent Document 5) were developed as methods for eliminating or reducing undifferentiated pluripotent stem cells from cell populations that underwent differentiation induction treatment. However, in the case of methods using a cell sorter, a large number of antibodies had to be used to recognize antigens present on the cell surface, thereby requiring considerable costs while also resulting in the problem of requiring a long period of time for treatment. In addition, methods incorporating suicide genes resulted in uncertainty regarding the safety of the cells incorporating such genes after transplant, thereby presenting problems in terms of clinical application. In addition, methods using chemical inhibitors had problems with efficiency and safety associated with the elimination or reduction of pluripotent stem cells.